Method for reducing analyzer variability using a normalization target

ABSTRACT

Disclosed herein is a method for improving the precision of a test result from an instrument with an optical system that detects a signal. The method comprises including in the instrument a normalization target disposed directly or indirectly in the optical path of the optical system. Also disclosed are instruments comprising a normalization target, and systems comprising such an instrument and a test device that receives a sample suspected of containing an analyte.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/975,686, filed May 9, 2018, now allowed, which is a continuation ofU.S. application Ser. No. 14/562,060, filed Dec. 5, 2014, now U.S. Pat.No. 9,989,466, issued on Jun. 5, 2018, which claims the benefit of U.S.Provisional Application No. 61/913,078, filed Dec. 6, 2013, each ofwhich is incorporated herein by reference in its entirety.

TECHNICAL FIELD

The subject matter described herein relates to systems, methods andapparatuses for improving the precision and reducing variability of anoptical analyzer, used in sample analysis to aid in medical diagnosis ordetection of the presence or absence of an analyte in the sample.

BACKGROUND

Assay devices, including detection gels, microfluidic devices,immunoassays, and the like, for detection of an analyte in a sample areknown in the art. In particular, lateral flow immunoassay devices areroutinely used for detecting the presence of an analyte in a sample.Lateral flow immunoassay devices often use a labeled specific-bindingreagent that is releasably immobilized on a test strip of porousmaterial. A liquid sample, such as a biological sample from a human oran environmental sample, is applied to an end of the porous strip andthe capillary properties of the strip transports the liquid sample alongthe strip, releasing the labeled specific binding reagent, which bindsspecifically to the analyte of interest at a first binding site thereof,if present, in the sample. The labeled binding reagent is then typicallycaptured at a test zone by a second reagent having specific binding fora second binding site of the analyte of interest. Excess labeled bindingreagent is captured at a control zone, downstream of the test zone by acontrol reagent which binds specifically to the labeled reagent.

Commercially available lateral flow assay devices are typically designedto be read by the naked eye of the user or to be read by an instrument.An instrument designed to read a signal emanating from a lateral flowdevice offers superior sensitivity relative to visually (i.e., nakedeye) read devices, as the optical system in an apparatus is able todetect intensities and wavelengths not visible to the naked eye.

A concern with instruments for reading lateral flow devices is the lackof precision, due to inter-instrument and/or operator variability. Thereremains a need in the art for an apparatus and a system that objectivelyanalyses a signal from an immunoassay test device, where the apparatushas improved sensitivity, accuracy and/or precision for determining thepresence or amount of signal from a lateral flow device.

The foregoing examples of the related art and limitations relatedtherewith are intended to be illustrative and not exclusive. Otherlimitations of the related art will become apparent to those of skill inthe art upon a reading of the specification and a study of the drawings.

BRIEF SUMMARY

The following aspects and embodiments thereof described and illustratedhereinbelow are meant to be exemplary and illustrative, not limiting inscope.

In some aspects, an apparatus for detection of a signal from a testdevice indicative of the presence or absence of an analyte in a sampleis provided.

In some aspects, a method to improve precision, accuracy or both of atest result from an optical instrument previously calibrated with anexternal calibrant is provided. The method comprises (a) assigning anormalization value to a normalization target embedded in or on theinstrument, the normalization value being assigned with respect to theprevious calibration, and storing the normalization value in instrumentmemory; (b) interrogating: (i) a test cartridge inserted into theinstrument to obtain a test signal; and (ii) the embedded normalizationtarget to obtain a current value each (the test cartridge and itstarget, as well as the embedded normalization target); and (c)optionally adjusting the test signal by a scaling factor, therebyobtaining a scaled test result.

In one embodiment, assigning a normalization value to the normalizationtarget comprises optically scanning the normalization target to obtain asignal from the normalization target or interrogating the normalizationtarget to obtain light output data. In one embodiment, opticallyscanning or interrogating the normalization target is conducted in asingle, continuous optical scan (or single data collection event) of thenormalization target and the external calibrant.

In one embodiment, assigning a normalization value to the normalizationtarget comprises optically gathering signal data by imaging thecalibrant and the normalization target in a single image to obtain asignal from the normalization target and the calibrant. In oneembodiment, the imaging system of the instrument obtains locationdependent light emission data from the test device and the normalizationtarget in a single data collection event.

In one embodiment, step (b) scanning comprises scanning in a single,continuous scan; that is, where the optical system initiates a passalong its optical pathway continuously and/or mechanicallyuninterrupted. That is, the imaging system of the instrument obtainslocation dependent light emission data from the test device and thenormalization target in a single data collection event. Scanning, in oneembodiment, contemplates securing an image using, for example, a CCD ora CMOS optical light gathering chips to image the test strip and thenormalization target in a single event.

In some aspects, a method to improve precision, accuracy or both of atest signal from an optical instrument is provided. The method cancomprise (a) providing an instrument pre-calibrated with an externalcalibrant, wherein the instrument comprises an embedded normalizationtarget; (b) collecting light emission data and assigning a normalizationvalue to the embedded normalization target, wherein the normalizationvalue is assigned with respect to the previous calibration, and whereinthe normalization value is stored in instrument memory; (c)interrogating: (i) a test cartridge inserted into the instrument toobtain a test signal; and (ii) the embedded normalization target toobtain a current value; and (d) optionally adjusting the test signal bya scaling factor, thereby obtaining a scaled test result with improvedprecision relative to the unadjusted test signal.

In one embodiment, step (c) interrogating comprises scanning or imagingin a single data collection event (e.g., a continuous scan where anoptical system initiates a pass along its optical pathway continuouslyand/or mechanically uninterrupted.)

In some aspects, a method and a system are provided for quantatitive orqualitative measurement of an analyte in a test sample. The system maycomprise an instrument with an optical pathway comprising (i) an opticsmodule or a fixed digital camera capable of interrogating a test stripcomprising the test sample when the test strip is positioned in theoptical pathway and (ii) an embedded normalization target affixed to theinstrument directly or indirectly in the optical pathway; and anexternal calibrant insertable into the instrument, directly orindirectly in the optical pathway.

In some embodiments, the test cartridge is scanned or interrogatedconcurrently with the embedded normalization target. In someembodiments, the test cartridge is scanned or interrogated subsequent tothe embedded normalization target.

In some embodiments, if the normalization value exceeds a predefinedvalue, a communication to a user is generated by the instrument. In someembodiments, if the assigned normalization value differs from thecurrent value by more than x %, the instrument issues a communication tothe user. In some embodiments, the communication advises that theinstrument requires re-calibration and/or normalization with theembedded normalization target. In some embodiments, the communicationadvises that an updated normalization value should be assigned to theembedded normalization target. In some embodiments, the value of x isabout 0.5%, 1%, 1.5%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%,15%, 18% or 20%.

In some embodiments, collecting light emission data comprisesinterrogating (or optically scanning) with an excitation wavelengthbetween about 300-800 nm, or between about 320-780 nm, or between about340-750 nm, or between about 340-800 nm, or between about 300-700 nm. Inother embodiments, the instrument comprises filters that cut offwavelengths less than about 300 nm and greater than about 500 nm so thetest device is not exposed to wavelengths that are below or above thesewavelengths.

In some embodiments, the scaling factor used to adjust the test signalis a ratio of the current value to the assigned normalization value. Insome embodiments, the instrument does not adjust the test signal by thescaling factor. In some embodiments, the calculation of the scaled valueof the test signal may be represented by the equation:ENT_(obs)/ENT_(NV)·test value=scaled value, where “ENT” represents theembedded normalization target, “obs” intends observed (the empiricalvalue); and “NV” denotes normalization value.

In order to prevent short-term bleaching of the embedded material due tofrequent scanning in a high throughput environment, the value assignedto the embedded calibrant may be date/time stamped and the instrumentmay issue an instruction to delay subsequent scans for a certain timeperiod. For example, in some embodiments, the drawer is not re-scanned(i.e., re-exposed to the UV excitation light) for 5 minutes to avoidphotobleaching. If an interval between scans is less than z seconds, thepeak height of the embedded material may not be updated for a subsequentscan. In some embodiments, z is about 300 seconds, about 180 seconds,about 120 seconds, about 60 seconds, about 45 seconds, or about 30seconds.

In some embodiments, the embedded normalization target and the externalcalibrant are constructed from the same material. In some embodiments,the embedded normalization target material comprises a plastic, plasticfilm, glass or fabric doped with an optical brightener. In someembodiments, the material is a plastic selected from the groupconsisting of polyester, polystyrene and polycarbonate. In someembodiments, the embedded normalization target material is a plasticcomprising an organic dye. In another embodiment, the embeddednormalization target is an epoxy or epoxy resin comprising a fluorescingcompound (i.e., a fluorophore or fluorescent chemical compound).

In some embodiments, the organic dye is a rhodamine. In someembodiments, rhodamine is selected from the group consisting ofrhodamine B, rhodamine 6G and sulphorhodamine G (SRG).

In some embodiments, the embedded normalization target is affixed in anoptical pathway in the instrument. In some embodiments, the embeddednormalization target is affixed to an element in the instrument that iscapable of receiving a test strip.

In some embodiments, the optical instrument comprises an optics modulefor detecting at least one of emitted fluorescent light or emittedreflected light. In another embodiment, the instrument comprises a fixeddigital camera like device, positioned such that light emitting from thetest device and normalization target is captured in a format that can beanalyzed by software in the instrument.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by reference to thedrawings and by study of the following descriptions.

Additional embodiments of the present methods, systems, apparatus, andthe like, will be apparent from the following description, drawings,examples, and claims. As can be appreciated from the foregoing andfollowing description, each and every feature described herein, and eachand every combination of two or more of such features, is includedwithin the scope of the present disclosure provided that the featuresincluded in such a combination are not mutually inconsistent. Inaddition, any feature or combination of features may be specificallyexcluded from any embodiment of the present disclosure. Additionalaspects and advantages of the present disclosure are set forth in thefollowing description and claims, particularly when considered inconjunction with the accompanying examples and drawings.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1C are front perspective (FIG. 1A), side (FIG. 1B) and top,partial (FIG. 1C) views of an apparatus, which comprises a drawer thatreceives a test device, the drawer shown in a closed position (FIG. 1A)and an open position (FIGS. 1B-1C);

FIG. 2 is a view of a calibration cassette;

FIG. 3 is an illustration of a test device wherein a test strip isenclosed in an optional housing sized for insertion into a drawer of anapparatus;

FIG. 4 is a schematic showing steps involved in calibrating anapparatus, and in detecting the presence or absence of an analyte in asample via test device inserted into an apparatus with a normalizationtarget; and

FIG. 5 shows the sequence of events in one embodiment of a measurementprocedure where an apparatus as described herein interacts with a testdevice, wherein the optical system of the apparatus scans the testdevice and a normalization target to report to a user the presence,absence and/or amount of analyte in a sample.

DETAILED DESCRIPTION I. Definitions

Various aspects now will be described more fully hereinafter. Suchaspects may, however, be embodied in many different forms and should notbe construed as limited to the embodiments set forth herein; rather,these embodiments are provided so that this disclosure will be thoroughand complete, and will fully convey its scope to those skilled in theart.

Where a range of values is provided, it is intended that eachintervening value between the upper and lower limit of that range andany other stated or intervening value in that stated range isencompassed within the disclosure. For example, if a range of 1 μm to 8μm is stated, it is intended that 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, and 7 μmare also explicitly disclosed, as well as the range of values greaterthan or equal to 1 μm and the range of values less than or equal to 8μm.

As used in this specification, the singular forms “a,” “an,” and “the”include plural referents unless the context clearly dictates otherwise.Thus, for example, reference to an “antibody” includes a single antibodyas well as two or more of the same or different antibodies, andreference to a “component” includes a single component as well as two ormore of the same or different components, and the like.

As used herein, “calibration” refers to the process of comparing ameasurement with a standard of known value (sometimes called a “goldstandard”) and adjusting the result/output measurement system of themeasuring instrument in accord with the standard. In some embodiments, acalibration cassette is generated, and calibrated against the goldstandard for use of the calibration cassette in an optical analyzer. Insome embodiments, the gold standard is fluorescent. In some embodiments,a fluorescent lanthanide target is used as a calibrant. In someembodiments, the fluorescent lanthanide is europium. In someembodiments, the external calibrant is rhodamine. In some embodiments, a“calibration value” is assigned to an external calibrant. In someembodiments, the calibration of an optical analyzer is recorded in theinstrument memory. In some embodiments, the calibration value of theinstrument represents an overall controlling value for subsequentmeasurements. In some embodiments, the instrument will be calibratedwithin each 2-30 day interval.

As used herein, “normalization” refers to a process disclosed herein, bywhich statistical error and instrument fluctuations are removed fromrepeated measurements. The present disclosure describes per scannormalization with an internal or “embedded normalizationtarget”/calibrant/reference material. In some embodiments, normalizationof the optical instrument refers to a value-assigned calibrationmaterial embedded within the instrument. In some embodiments, a“normalization value” is assigned to an embedded normalization target.In some embodiments, the embedded normalization target is fluorescent.In some embodiments, a fluorescent lanthanide or a fluorescent organicdye, such as rhodamine, target is used as a calibrant. In someembodiments, a normalization target value is assigned on a per scanbasis. In some embodiments, the normalization value will be reset ateach calibration event. A significant change in normalization value fromone scan to the next may be flagged as an error and a communication issent to the user of the instrument.

Accuracy is the degree of closeness of measurements of a quantity to anactual true value.

Precision is the degree to which repeated measurements under unchangedconditions show the same result.

In some embodiments, “assigning a value to an embedded normalizationtarget based on an external calibration value” means to relate anexternally calibrated value based on a gold standard with an opticallydetermined value from the embedded normalization target. The processdisclosed herein allows the dismissal of errors due to inter-instrumenterrors, intra-instrument or user errors, and outliers from optical scansand resulting data. With regard to errors based on inter-instrumentdifferences, the external calibrant assures that each instrumentresponds within a certain percentage (e.g., about 1%, 2%, 3%, 4%, 5%) ofsignal from the external calibrant and the embedded normalization targetattenuates variability in readings for a specific individual instrument.In some embodiments, a scan or image results in capturing peak heightdata. In some embodiments, data collection after per-scan or per-imagenormalization eliminates outliers near the limits of the repeatabilityspecification. In some embodiments, per-scan/per-image normalization isnot necessary for analyzers demonstrating excellent repeatability. Insome embodiments, the presently disclosed methods, systems, analyzersand the like reduce variability to less than system performancespecifications without normalization capabilities. In one embodiment,the variability is reduced, for example, by at least about 2%, 3%, 4%,5%, 8%, 10%, 15%, 20% or 25%.

In some embodiments, assigning a normalization value to an embeddednormalization target allows reduction of instrument inter- andintra-variability. In some embodiments, assigning a normalization valueto an embedded normalization target allows reduction of “noise,” definedas a non-specific, inaccurate and/or imprecise signal. In someembodiments, assigning a normalization value to an embeddednormalization target allows the user of an optical analyzer to improve asignal to noise ratio.

“Stable” means that a signal from a standard or target being measureddoes not degrade in spite of multiple scans. A stable fluorescent targetmay be embedded in the optical analyzer, for example in a drawer thatreceives a test strip.

“Scanning in a single scan” means that the optics module scans/reads thenormalization target and the test strip in one continuous pass of theoptics module along its optical pathway. In some embodiments, the opticsmodule scans/reads the embedded normalization target first and the teststrip second in the single scan. In some embodiments, the optics modulescans/reads the test strip first and the embedded normalization targetsecond in the single scan.

“Imaging a test strip” means that a digital camera like device using aCCD or CMOS light sensor collects light output data from the test in away that can be processed into signal data by software in theinstrument.

Systems and apparatuses for analysis of samples to aid in medicaldiagnosis and/or detection of the presence or absence of an analyte in asample are also disclosed in U.S. Patent Application Publication No.2013/0230844 and U.S. Patent Application Publication No. 2013/0230845,the disclosure of each incorporated herein by reference herein in itsentirety.

Also of interest are the following patents and applications: U.S. Pat.No. 7,521,260, issued 21 Apr. 2009, describing diagnostic test stripsand systems for optically reading them that employ “a data analyzer thatperforms at least one of (a) identifying ones of the light intensitymeasurements obtained from the test region based on at least onemeasurement obtained from the at least one reference feature, and (b)generating a control signal modifying at least one operational parameterof the reader based on at least one measurement obtained from the atleast one reference feature; U.S. Pat. No. 7,925,445, issued 12 Apr.2011, describing a read-write assay system; U.S. Pat. No. 8,334,522,issued 18 Dec. 2012, describing optical methods and apparatuses forquantitatively determining the concentration of fluorophores of asubstance in a sample; and U.S. Patent Application Publication2012/0300205 describing a method of normalizing a fluorescence analyzerinvolving multiple measuring steps.

II. Method to Improve Instrument Precision

In a first aspect, a method for improving the precision of a test resultfrom an instrument that optically detects a signal from a test device isprovided. The method, in other embodiments, improves accuracy of a testresult. The method, in other embodiments, improves both precision andaccuracy of a test result. The method(s) comprise providing aninstrument which comprises a normalization target embedded in or on theinstrument, directly or indirectly in the pathway of the optical systemof the instrument. A signal from the normalization target is obtainedeach time a signal from a test device is obtained, and the current valueof the normalization target signal is compared to a stored value of thenormalization target. The signal from the test device can be adjusted orscaled, as needed, based on the current value and stored value of thenormalization target. The method will be described in more detail below,and examples of improved precision of test results illustrated. First,however, an exemplary optical instrument (also referred to herein as anapparatus) and exemplary test device are described. In the descriptionbelow, the test device is exemplified by a lateral flow immunoassay, andis sometimes referred to as a test strip. It will be appreciated thatthe test device is not intended to be limited to the lateral flowimmunoassay test device used to exemplify the system, and a skilledartisan will appreciate that other test devices, such as microfluidicdevices, immunoassays other than lateral flow based immunoassays, arecontemplated.

Apparatus

An exemplary apparatus contemplated for use in the method herein isdescribed in U.S. Patent Application Publication No. 2013/0230844 and inU.S. Patent Application Publication No. 2013/0230845, each of which isincorporated by reference herein in its entirety. FIGS. 1A-1C are viewsof an exemplary apparatus, which will not be briefly described.

FIG. 1A is a perspective view of apparatus 10 which includes a housing12 that encloses an optics system, electronics software, and othercomponents of the apparatus. A front side 14 of the apparatus includes auser interface 16 that may include, for example, a key pad 18 and adisplay screen 20. The apparatus also includes a drawer 32 movablebetween open and closed positions, as shown in FIG. 1A in its closedposition and in FIGS. 1B-1C in its open position. In the embodimentshown, the drawer is positioned on a front edge 34 of the apparatus. Itwill be appreciated that the drawer could also be positioned on eitherside of the apparatus. The drawer can move between its open and closedpositions by a mechanical mechanism, such as a latch and springmechanism, or in response to a user activating a key on the front orface of the apparatus, such as an “open drawer” or “eject test device”button. In one embodiment, the drawer is moved into its closed positionafter insertion of a test device manually by a user, or in response to auser activating a key or button on the apparatus. The drawer isconfigured to receive an immunoassay test device, such as test device36, further described below. Within the drawer, in one embodiment, is adistinct region, for example a depression, sized to receive the testdevice. During operation of the apparatus, the test device remains in astationary position in the drawer, and therefore is positioned withprecision in the apparatus for precise interaction with a movable opticsmodule.

The apparatus also includes a normalization target disposed in or on theapparatus. The normalization target, in a preferred embodiment, isdirectly in the optical pathway of the optics module (described below),and an exemplary position of the normalization target 40 is indicted inFIG. 1B. Normalization target 40, in one embodiment, is disposed orembedded on drawer 32, and in a position that remains exposed to theoptics module when a test device is inserted in the drawer. It will beappreciated that the normalization target 40 can also be disposed orembedded in the instrument indirectly in the optical pathway, such as ona side wall or positioning arm of the instrument, and mirrors or othertechniques can be used to achieve interaction of the normalizationtarget with the optics module. The normalization target generally takesthe form of a material that is deposited on the instrument or embeddedin the instrument in a position for interaction with the optical system.The material is one that responds to a source of excitation light in theoptics system by emitting energy in the form of signal detectable by aphotodetector in the optics system. Exemplary materials are discussedbelow.

In general, the apparatus will comprise a drawer movable between an openposition and a closed position where the drawer is contained within thehousing; an optional bar code scanner positioned in the housing forreading an encoded label on a test assay (or test device) to be insertedinto the apparatus; a carriage movably mounted in the housing, thecarriage comprising a source of excitation light and a photodetector fordetecting energy emitted; drive electronics to move the carriagesequentially from a first position to a final position, and a pluralityof positions there between, wherein the carriage has a dwell time ateach of said plurality of positions between the first and finalpositions; a processor for control of the apparatus, wherein theprocessor comprises a software program that processes data obtained fromthe photodetector by generating a data array comprised of emitted signalat each incremental position between the first and final positions,taking the first derivative of the data array to form a derivative dataset, wherein a first maximal value in the derivative data setcorresponds to a maximum signal from a reference line or a control lineon the test device, and the position in the derivate data array of thefirst maximal value determines the position of data from theanalyte-specific test line.

In some embodiments, when the instrument is first powered on, thesoftware runs a check for the time and date stamp of the normalizationvalue stored in memory that was assigned to the embedded normalizationtarget, and (i) if an assigned normalization value is found, and it isless than a certain age, this assigned normalization value is used foradjusting (or scaling) a test signal to be obtained, or (ii) if anassigned normalization value is not found in memory, or if thepreviously assigned normalization value is of a certain age, the user isinstructed to proceed to scan and assign a new or updated normalizationvalue to the embedded normalization target. In other embodiments, if anassigned normalization target value changes more than a pre-definedamount relative to an assigned normalization value stored in memory,then a communication is sent to the user, which may indicate that theinstrument drawer should be removed and cleaned, or that the newlyassigned normalization value is beyond a certain limit with respect tothe external calibrant, and/or that the instrument should berecalibrated with a gold standard. In some embodiments, there may be nocommunication to the user, and the instrument may enter a lock-downmode, forcing recalibration and/or renormalization. In some embodiments,a barcode inside the instrument may be present which correlates with thenormalization target such that the instrument will recognize differentdrawers or normalization targets.

As mentioned above, the apparatus includes a movable optics system inthe apparatus, to scan the stationary test device inserted into theapparatus. The microprocessor-controlled optics system is positionedwithin the housing of the apparatus such that it moves along thelongitudinal axis (along the line denoted z-z in FIG. 1A) from a home orstart position to a final position. The optics system includes an opticsmodule comprised of a carriage mounted on a track (e.g., lead screw),the carriage movable by an electric motor or actuator within the opticssystem of the apparatus. Secured to the carriage, and part of themovable optics module, are an illumination source and a detector, suchas a photodiode. The illumination source is typically mounted toilluminate the test device, for example perpendicular to the testdevice, and the detector oriented at an angle to collect emission fromthe test device. In some embodiments, a photodiode is oriented at 40°relative to the test device, and more generally the detector can beoriented at an angle of between about 20°-75° relative to the surface ofthe test device. In some embodiments, the optics module includes asingle element optical detector (that is, an array of optical detectorsis not present) and a single illumination source. The optics module canalso comprise one or more filters, and can include a filter, in someinstances a long pass filter, on the emission side of the illuminationsource, and a filter positioned between the test device and thedetector. In some embodiments, the illumination source emits UV light ata wavelength that matches the excitation wavelength of a label in thetest device. In some embodiments, the illumination source is a lightemitting diode (LED) that has a peak emission at 365 nm, more generallyof between about 320-390 nm or 325-380 nm. In this embodiment, the bandpass or interference filter positioned in the optical path from the LEDto the test device transmits light between 340-400 nm or between 310-315nm.

In some embodiments, the photodetector is a broad band detector suitableto detect light at the wavelength emitted from the label in the testdevice. In some embodiments, the photodetector is a single-elementphotodetector (i.e., is not an array of photodetectors). In someembodiments, the label is or contains a fluorescent, luminescent,chemiluminescent compound. As will be described below, an exemplaryfluorescent label is a lanthanide ion, such as europium, samarium,terbium and holmium, which each fluoresce at specific wavelengths. Afilter may be positioned in the optical path between the test device andthe detector, in some embodiments, and can transmit light above about515 nm for detection by the detector. A skilled artisan will appreciatethat a variety of filters are known in the art (longpass, shortpass,bandpass, etc.) and can be selected according to the wavelengths oflight desired to excite a label and the wavelength of light desired fordetection.

The apparatus, in some embodiments, includes a temperature sensingmeans, and in some embodiments, includes at least two temperaturesensing devices housed within the housing of the apparatus. A firstinternal temperature sensor is positioned to detect the temperature inthe region associated with the optics system and a second internaltemperature sensor is positioned elsewhere in the apparatus away fromany internally generated heat source in order to detect ambienttemperature of the environment in which the apparatus is operated.

The apparatus includes internal memory storage with necessary softwarefor operation and for storage of data collected from sample analysis. Bythe SIM port or by an external computer (wireless or wired attachment),data can be exported from the apparatus or imported to the apparatus.

As mentioned above, the apparatus may be equipped with ports forattachment to optional external devices. An external bar code scannermay be attached to the apparatus. Such a bar code scanner interfaceswith the apparatus via a suitable data port provided on the apparatus.Externally attached devices ease transfer of data into and from theapparatus, and can eliminate user keyboard input, permitting accuratedata input into the apparatus regarding a test to be analyzed or patientor sample information. In some embodiments, a barcode scanner externalis attachable via PS-2 port on the apparatus and is capable of reading alinear or 1D bar code. In some embodiments, the apparatus is wireless orwired connected to a device for delivering data to a third party.

External Calibrant and Normalization Target

As described above, the apparatus comprises an optics system comprisedof an optics module that includes a light source, which in someembodiments is an LED light source with a peak emission at 365 nm. Thedetector for the emitted signal, such as fluorescent light from a labelin a test strip, is a photodiode with filters to ensure that the lightfrom the fluorescent reagent is not contaminated by ambient norexcitation light. Signal from the photodiode is translated through ananalog to a digital converter where the digital signal is processed by amicroprocessor in the apparatus into a test result. To ensure consistentlight output, the LED has a feedback loop whereby the optics systemmonitors the light output of the LED and triggers an adjustment of theelectrical current to the LED to ensure a consistent intensity of theexcitation light beam in real time. To further ensure that signal driftis controlled, the apparatus has a calibration algorithm that enablesthe user to insert an external calibrant specifically designed for theapparatus and provided with the apparatus. The apparatus also includes anormalization target positioned for interaction with the optics system,where signal from the normalization target is detected each time signalis detected from a test device inserted into the instrument fordetection of an analyte.

In another embodiment, the apparatus comprises, rather than an opticsmodule, a fixed digital camera like device. The imaging device islocated such that light emitting from the test device is captured in aformat that can be analyzed by software in the apparatus.

An exemplary external calibrant, in the form of a calibration cassette,is illustrated in FIG. 2. Calibration cassette 70 comprised of acalibration strip 72 secured within an optional housing member, such ashousing member 74 which is separable in this embodiment into uppermember 74 a and lower member 74 b. A window 76 in upper housing member74 a is provided so that the optics system in the apparatus can interactwith one or more lines on the calibration strip. The calibration stripcan comprise one or more lines, and in various embodiments, comprisestwo or more lines, three or more lines or four or more lines. In someembodiments, the calibration strip comprises at least two lines, atleast three lines, or at least four lines. The embodiment illustrated inFIG. 2 shows a calibration strip with four lines, identified as 78, 80,82 and 84, and referred to herein below as calibration lines orcalibration test lines. The calibration lines are positioned on thestrip relative to the housing to be visible through the window when thestrip is secured within the housing.

In some embodiments, the normalization target and/or calibration stripis/are comprised of a material that fluoresces upon excitation by lightfrom the illumination source in the optics system of the apparatus at awavelength detectable by the photodiode subsequent to passage throughany filter(s) in the light path of the photodiode. In some embodiments,the normalization target and/or calibration strip is comprised of amaterial that fluoresces, and the calibration lines or normalizationtarget are defined by masking. For example, the fluorescing material canbe silk-screened with a material that blocks light entering and leavingthe one or more calibration lines exposed. Alternatively, a fluorescingmaterial can be deposited in discrete lines onto a non-fluorescingmaterial to form the normalization target or calibration lines of theexternal calibrant.

In some embodiments, the fluorescing material is a fluorescent whiteningagent, also known as fluorescence brightners, deposited on or dispersedin a support material. Exemplary fluorescent whitening agents opticalbrightener are dyes that absorb light in generally the ultraviolet andviolet range (340-370 nm) of the electromagnetic spectrum and re-emitlight in the blue region (typically 420-470 nm). Exemplary opticalbrighteners include compounds such as stilbenes (di-, tetra, orhexa-sulfonated), coumarins, imidazolines, diazoles, triazoles,benzoxazolines, biphenyl-stilbenes. A specific exemplary class ofcompounds are thiophenediyl benzoxazole compounds, and a specificexemplary fluorescent whitening agent is2,5-thiophenediylbis(5-tert-butyl-1,3-benzoxazole), an opticalbrightener. Exemplary support materials include polymers, andparticularly plastics, such as polymethylmethacrylates and polyesters,in particular biaxially oriented polyester. The whitening agent can bepolymerized with the support material during manufacture of thepolymeric support material, or can be deposited onto the polymericsupport after its manufacture. In some embodiments, the fluorescingmaterial forming the one or more calibration lines or normalizationtarget fluoresces between 500-650 nm when excited.

The calibration cassette can optionally include a label, such as barcode 86 on the cassette in FIG. 2. In some embodiments, the bar code isa linear or a two-dimensional bar code with information, for example, toconfirm for the apparatus that the cassette is a calibration cassetteand with information regarding an expiration date for the cassette.

The calibration cassette is dimensioned to fit within the drawer of theapparatus, for interaction with the optics system. The calibrationcassette when inserted into the instrument does not block thenormalization target that is deposited in or embedded internally in theinstrument for interaction with the optic system, so that during whenthe optics system moves in a single, continuous scan from its first tofinal position, it interacts with both the external calibrant and thenormalization target. Signal emitted from the external calibrant and thenormalization target are detected and stored in instrument memory.

A user of the apparatus, typically when prompted by the apparatus at aregular, defined period, such as every 30 days, or once a month, or onceevery two months, etc., inserts the external calibrant/calibrationcassette into the drawer of the apparatus. The internal bar code readerwithin the apparatus transfers the information on the barcode of thecalibration cassette to the processor in the analyzer. It will beappreciated that the internal bar code reader is an optional feature, asthe information on the bar code label can be entered into the apparatusby a user using the key pad or via an external bar code scanner. Fromthis information, the instrument will confirm that a calibrationcassette has been inserted into the analyzer, provide target signals theinstrument uses for comparison to actual signals obtained for thecalibration lines on the calibration strip, and provide the expirationdate of the calibration cassette. The instrument then activates theoptics system to initiate illumination of the calibration cassette, andspecifically sequential illumination of each of the calibration linesvisible within the calibration cassette window. The instrument thendetects the fluorescent signal from each of the calibration lines andstores the signal in memory. The instrument also detected thefluorescent signal from the normalization target embedded in or on theinstrument.

With regard to the signal from the external calibrant, the detectedsignal for each of two of the calibration lines from the externalcalibrant is compared to the expected signal (e.g., a signal valuestored in memory or provided on the bar code of the external calibrant)for that calibration line. If the detected signal for each of two of thecalibration lines is within a predefined range of the target signal, forexample within (+/−) 1.75, 2%, 2.25%, 2.5% or 3%, then the calibrationof the analyzer is valid and no adjustments to the apparatus are needed.This calibration check event is recorded and stored in the memory of theapparatus. If the detected signal for either or both of two of thecalibration lines of the external calibrant is outside the predefinedrange of the target signal, but not outside of a maximum predefinedrange, for example outside +/−3.25%, +/−3.5%, +/−3.75% or +/−4% of apredefined target signal for a specific line, the processer in theanalyzer activates an algorithm to self-calibrate using a third ordifferent calibration line on the calibration test strip. Informationfor the expected signal from this third line is also in the barcodeinformation and was conveyed to the apparatus upon insertion of thecalibration cassette and scanning of the bar code. When the signal forthis third calibration line is within a defined acceptable range, theanalyzer again reads the first two calibration lines to confirm that theexpected target signal is detected for these two lines. If the signal isoutside the maximum range, the analyzer cannot recalibrate itself, andthe system generates an error message that is displayed to the user.

With regard to the signal from the normalization target, in embodimentswhere the material of the normalization target is identical to thematerial of the external calibrant, a normalization value is assigned tothe normalization target based on the signal from the externalcalibrant. In embodiments where the material of the normalization targetis different from the material of the external calibrant, anormalization value is assigned to the normalization target based onsignal from the normalization target that is adjusted by a factor storedin instrument memory that accounts for expected signal differencesbetween the external calibrant material and the normalization targetmaterial. The normalization value assigned to the normalization targetis stored in instrument memory.

Test Device: Exemplary Lateral Flow Immunoassay

The apparatus described above is designed to receive and interact with atest device, and an example of a test device is shown in FIG. 3. In FIG.3, a test device comprising a test strip enclosed in an optional housingthat is sized for insertion into a drawer of an apparatus is shown. Thetest device is exemplified in the drawings below by a lateral flow testimmunoassay, however it will be appreciated that a lateral flowimmunoassay is exemplary of test devices suitable for interaction withthe apparatus.

Test device 100 is comprised of, in sequence, a sample pad 102, a labelpad 104, one or more lines indicated collectively at 106 and selectedfrom a test line, a control line and a reference line, and an absorbentpad 108. In some embodiments, a support member 110 is provided, and eachor some of the sample pad, label pad, lines and absorbent pad aredisposed on the support member. In some embodiments, the test deviceadditionally includes a desiccant portion, which can be positioned onthe support member of the test device, and in some embodiments isdisposed on the support member downstream of the absorbent pad, asdescribed in U.S. Patent Application Publication No. 2008/0311002,incorporated by reference herein, or can take the form of a discretecomponent, physically separate from the test strip, inserted into ahousing member that contains the test strip, such as desiccant 112.

In some embodiments, the test strip is enclosed in an optional housing,sometimes referred to herein as a cassette. The optional housing may becomprised of an upper member 116 and a lower member 118 that fittogether to form a housing. The lower member may include architecturalfeatures that define dimensioned regions for receiving the test stripand an optional desiccant. The upper housing member includes at leasttwo openings, a first sample input port 120 and a viewing window 122.The sample input port is disposed directly above the sample pad on thetest strip, so that a sample dispensed into the sample input portcontact the sample pad for flow along the test strip. The sample inputport can include a bowl portion to receive a liquid sample into theport. The viewing window is positioned to reveal the lines in the teststrip, so the optics system in the apparatus can interact with thelines.

In some embodiments, a bar code label is affixed to the upper housingmember. It will be appreciated that the bar code label can be positionedelsewhere on the housing, and is positioned for interaction with theinternal bar code scanner positioned within the apparatus. In someembodiments, the bar code label is a 2D bar code, encoding information,for example, regarding the assay test strip, such as thepathogen/analyte the test strip is designed to detect (Flu A/B, Strep A,RSV, others listed below, etc.) which informs the apparatus whatprotocol in memory to initiate for scanning the test strip; a uniquetest serial number so that the apparatus will not read same test striptwice. In some embodiments, the information contained in the bar codedoes not include information related to the patient or the sample type,and is limited to information about the test strip.

It will be appreciated that the test device illustrated in FIG. 3 isexemplary of lateral flow test devices in general. The test strip can beconfigured uniquely for any given analyte, and the external housing isoptional, and if present, need not be a cassette housing but can be aflexible laminate, such as that disclosed in U.S. Patent ApplicationPublication No. 2009/02263854 and shown in Design patent No. D606664,both of which are incorporated by reference herein. The system is suchthat the drawer in the apparatus and the test device are dimensioned toreceive the test device in the drawer, and the optics system in theapparatus has a movement path the scans the necessary regions of thetest device.

In that regard, an exemplary test strip may include a sample receivingzone in fluid communication with a label zone. A fluid sample placed onor in the sample zone flows by capillary action from the sample zone ina downstream direction. A label zone is in fluid communication with atleast a test line and a control line or a reference line. In someembodiments, the label zone is in fluid communication with a negativecontrol line, an analyte test line, an optional second analyte testline, and a reference line. The two or more lines are in fluidcommunication with an absorbent zone. That is, the label zone isdownstream from the sample zone, and the series of control and testlines are downstream from the label zone, and the absorbent pad isdownstream from the portion of the test strip on which the lines arepositioned. A region between the downstream edge of the most downstreamanalyte-specific test line and the upstream edge of the absorbent pad isa procedural control zone. A reference line is within the proceduralcontrol zone. As will be described below, the procedural control zone,and in particular the reference line therein, (i) ascertains whethersample flow along the test strip occurred based on its RLU signal(emission), and (ii) may be used by the analyzer (or more properly, analgorithm stored within the analyzer) to determine the relativelocations of the other lines (control, if present, and analyte-specifictest line(s)) on the test strip. The reference line may also be used toascertain a cut-off value, as will be described below, to render theimmunoassay insensitive to incubation time, and in particularinsensitive to incubation time over a period of 1-15 minutes, a periodof 1-12 minutes, a period of 1-10 minutes or a period of 2-10 minutes.Materials for construction of each of the zones is well known in theart, and includes, for example, a glass fiber material for the samplezone, a nitrocellulose material on which the two or more lines arepositioned.

The sample zone receives the sample suspected of containing an analyteof interest control its flow into the label zone. The label zone, insome embodiments, contains two dried conjugates that are comprised ofparticles containing a lanthanide element. The lanthanide materialsinclude the fifteen elements lanthanum, cerium, praseodymium, neodymium,promethium, samarium, europium, gadolinium, terbium, dysprosium,holmium, erbium, ytterbium, lutetium, and yttrium. In some embodiments,the dried conjugates are particles which are polystyrene particles ormicroparticles (particles less than about 1,000 micrometers in diameter,in some instances less than about 500 micrometers in diameter, in someinstances less than 200, 150 or 100 micrometers in diameter) containinga luminescent or fluorescent lanthanide, wherein in some embodiments,the lanthanide is europium. In some embodiments, the lanthanide is achelated europium. The microparticles, in some embodiments, have a coreof a lanthanide material with a polymeric coating, such as an europiumcore with polystyrene coating. A binding partner for the analyte(s) ofinterest in the sample is/are attached to or associated with the outersurface of the microparticles. In some embodiments, the binding partnerfor the analyte(s) of interest is an antibody, a monoclonal antibody ora polyclonal antibody. A skilled artisan will appreciate that otherbinding partners can be selected, and can include complexes such as abiotin and strepavidin complex. Upon entering the label zone, the liquidsample hydrates, suspends and mobilizes the dried microparticle-antibodyconjugates and carries the conjugates together with the sampledownstream on the test strip to the control or reference and test linesdisposed on the nitrocellulose strip. If an analyte of interest ispresent in the sample, it will bind to its respective conjugate as thespecimen and microparticles flow from the label zone onto the surface ofthe nitrocellulose. In some embodiments, this flowing mixture will thenencounter negative control line. The negative control line may becomprised of mouse immunoglobulin (IgG) to enable detection ofnon-specific binding of the conjugates to the immunoglobulin, thusapproximating the level of non-specific binding that will occur at thedownstream test line(s). The signal generated at this negative controlline is used to help ensure that high non-specific binding at theanalyte-specific test line does not lead to false positive results.

For example, as the sample and microparticle-antibody conjugatescontinue to flow downstream, if the analyte of interest is present inthe sample, the fluorescent microparticle-antibody conjugate, which isnow bound with antigen/analyte of interest, will bind to the specificbinding member for the analyte of interest that is immobilized at thetest line(s). In some embodiments, a single test line is present on thetest strip. In some embodiments, at least two, or two or more test linesare present on the strip. By way of example, a test strip intended fordetection and/or discrimination of influenza A and influenza B caninclude a first test line to detect influenza A and a second test lineto detect influenza B. Microparticle-antibody conjugates comprised ofmicroparticles coated with antibodies specific for influenza A andmicroparticles coated with antibodies specific for influenza B may beincluded in the label zone, and in some embodiments, downstream of thenegative control line. A first test line for influenza A and a secondtest line for influenza B can be disposed downstream of the label zone.The first test line for influenza A comprises a monoclonal or polyclonalantibody to a determinant on the nucleoprotein of influenza A and thesecond test line for influenza B comprises a monoclonal or polyclonalantibody to a determinant on the nucleoprotein of influenza B. Ifantigen is present in the sample, a typical immunoassay sandwich willform on the respective test line that matches the antigen in the sample.

The microparticle-antibody conjugates that do not bind to the negativecontrol line or to a test line continue to flow by capillary actiondownstream, and the remaining sample encounters the reference line, insome embodiments proceeding into the absorbent pad.

Test Procedure and Instrument Operation

FIG. 4 is a flow chart showing steps involved in calibrating anapparatus using an external calibrant, and in detecting the presence orabsence of an analyte in a sample via test device inserted into anapparatus with a normalization target. In step 00, the externalcalibrant is calibrated using a recognized measurement standard (oftenreferred to in the industry as a ‘gold standard’). Instruments thatdemonstrate a signal output for the external calibrant material that isat or within a fixed range of the mean signal from a manufacturing lotof instruments are selected to serve as the ‘gold’ or calibrationinstruments. These gold instruments are used to assign a value to theexternal calibrant.

Once the external calibrant has a value assigned to it based on a goldstandard, the external calibrant is used to calibrate a specificinstrument. The external calibrant, as denoted in Step 0 of FIG. 4, isinserted into the instrument, signal from the external calibrant isdetected upon excitation by the optics system, and the detected signalcompared to the expected value of the external calibrant, which in someembodiments can be a value stored on a barcode on the external calibrantthat is read by the instrument. By way of example, and with reference tothe exemplary external calibration cassette of FIG. 2, if the observedvalues from line 1 and line 4 (peak 1 and peak 4) are within apre-determined range (e.g., 2%) of the expected external calibrant valuethe instrument is deemed to be calibrated, and need not be recalibrated.If either of the observed values is greater than the pre-determinedrange (e.g., 2%) but less than a certain amount, e.g., 10%, differentfrom the expected calibrant value then the instrument recalibrates basedon the difference between the observed value for line 3/peak 3 versusthe expected external calibrant value for line 3/peak 3. The instrumentcalibration factor is changed according to the line 3/peak 3 measurementand is stored in instrument memory as the external calibrant value.

Next, with the external calibrant inserted into the machine, the opticalsystem excites the external calibrant and the instrument-embeddednormalization target. Signal emitted from the normalization target isdetected and signal from the external calibrant is detected, the signalspreferably obtained in a single scan of the optical system as it movesuninterrupted along the optical pathway. A normalization value isassigned to the normalization target, based on the detected signal forthe normalization target and the detected signal of the externalcalibrant, as indicated in Step 1 of FIG. 4. The normalization value ofthe normalization target is stored in instrument memory. A comparison ismade of the percent difference of the external calibrant signal (peakheight) in comparison to the signal (peak heights) from thenormalization target observed versus stored value. If this relativedifference is larger than a preset value, the user is prompted to reruncalibration with the external calibrant, clean the normalization target,or replace the external calibrant.

With continued reference to FIG. 4, a test device comprising a samplesuspected of containing an analyte is provided, and the test device withthe sample is inserted into the instrument. An optical scan of the testdevice is initiated by the user, and additional details on the sequenceof events that occur within the instrument upon initiation of a testscan of a test device are provided in FIG. 5. As denoted in Step 2 ofFIG. 4, the optical system of the instrument scans the test device andthe embedded normalization target, preferably in a single, continuous,uninterrupted scan of the optics module from its start position to itsfinal position. Signal emitted from the normalization target isdetected, as is signal from the test line(s), control and referencelines (if present) on the test device. If the measured signal from thenormalization target deviates by x % or more from the assignednormalization value stored in instrument memory, the instrument isprogrammed to communicate to the user that the instrument may requirerenormalization (Step 3 a, FIG. 4). In this situation, a user wouldremove the test device and insert the external calibrant and initiationa scan of the optics system, to assign an updated normalization value tothe normalization target based on a scan of the external calibrant andthe normalization target (e.g., Step 1 of FIG. 4 is performed). Thevalue of x can be present in the instrument software, and a typicalvalue of x is 0.5%, 1%, 1.5%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%,14%, 15%, 18% or 20%. The software of the instrument also determines thetime lapse between the current scan and the time since the last orprevious scan of the normalization target. If the time since the lastscan of the normalization target is less than or equal to z seconds, theinstrument is programmed to use the normalization value from the priorscan of the embedded normalization target (Step 3 b, FIG. 4). Thisoptional feature is to avoid effects from photobleaching of thenormalization target. The value of z will depend on the material of thenormalization target, and is a value that can be programmed into thesoftware of the instrument or provided on a bar code external to theinstrument. The value of z can be, for example, between 5-360 seconds,or 10-300 seconds, or 15-300 seconds, or 30-360 seconds. In anotherembodiment, a the value of z is less than or equal to 300 seconds, 180seconds, 120 seconds, 60 seconds, 45 seconds, or 30 seconds.

Signal detected from the one or more lines on the test device may beadjusted by a scaling factor that is determined from the scannednormalization value and the assigned normalization value (stored ininstrument memory), as indicated in Step 4 of FIG. 4. In someembodiments, the scaling factor may be equal to the ratio of the scannednormalization target value gained from Step 2 to the normalization valueassigned in Step 1.

FIG. 5 shows the sequence of events in one embodiment of a measurementprocedure where an apparatus as described herein interacts with a testdevice, wherein the optical system of the apparatus scans the testdevice and a normalization target to report to a user the presence,absence and/or amount of analyte in a sample. To initiate a scan of atest device, the apparatus is powered-on if needed and the toggle switchto initiate the apparatus software is activated. Prior to inserting thetest device with sample into the apparatus, using the optional externalbar code reader information about the user, the sample, the patient,etc. can be scanned into the apparatus memory. With reference to FIG. 6,a “start test” button on the apparatus or on the touch screen ispressed, 150, to start a measurement of a test device. The apparatustakes an optional temperature reading, 152, and then automatically opensthe drawer in the apparatus, 154, to receive the test device on which asample has been dispensed onto the sample pad. The test device withloaded sample is inserted into the drawer, 156, and the drawer is closedmanually, 158, with gentle pressure by the user. As the drawer closes,one or more positioning arms press against the test device to positionit in the drawer in a precise location that is consistent from test totest. The optics shield within the apparatus is positioned to protectthe optics system and its movable optics module from any liquid samplethat may splash from the sample input port when the drawer closes.

Closure of the drawer initiates a sequence of events, 158, comprised ofthe following: The internal bar code reader scans the bar code on thetest device and receives information regarding the assay type (e.g.,influenza A/B, hCG, Strep A, RSV, etc.), the serial number and theexpiration date of the test device, optical cut-off information for theassay type, and any other information included on the bar code securedto the test device. In some embodiments, a mirror is positioned tofacilitate interaction of the light beam from the internal bar codescanner and the bar code label on the test device. It will beappreciated that the internal bar code reader is an optional feature, asthe information on the bar code label can be entered into the apparatusby a user using the key pad or via an external bar code scanner. Basedon the test assay type discerned from the information on the bar codelabel or otherwise provided to the apparatus processor, the apparatusinitiates an algorithm stored in the apparatus's memory for the assayfor which the test device is designed, and based on user definedselection of read-now mode or walk-away mode, a protocol stored inmemory initiates. In walk-away mode the apparatus incubates for a periodof time, 160, prior to initiating a scan of the test device, 162; inread-now mode the apparatus does not wait for the preset incubation timefor that particular assay, and immediately begins a scan of the testdevice, 162.

The scan and evaluation of the test device and the embeddednormalization target, 162, comprises another optional temperature check,164, at the same or different position from the first temperature check152. The initiated algorithm activates the optics system, including thestepper motor that moves the optics module with respect to the testdevice that is stationary in the apparatus. The optics system searchesfor its home position, 166, (described below) and then conducts a scanof the measurement window, 168, in the housing of the test devicethrough which the reference/control and test line(s) are visible. Thisscan also includes a scan of the normalization target. The motor in theoptics system moves the optics module incrementally along an opticalpathway between a start position and to a final position; the opticalpathway including the normalization target and the length of themeasurement window in the test device in accord with parameters definedby the algorithm for the particular assay being conducted. The opticsmodule is moved in incremental steps by the motor in the optics systemalong the optical pathway, typically in a downstream to upstreamdirection with respect to sample fluid flow on the test strip, whereinthe optics carriage stops at each incremental step or position toilluminate the position, and detect emitted light after illumination atthat position, before advancing upstream to the next position.

After collection of emitted light at each of the plurality ofincremental positions along the length of the test window, the algorithmlocates and evaluates the data in the data array that is associated withthe signal emitted from the normalization target, the test lines,control and reference lines (if present), 170, and conducts aqualitative, semi-quantitative or quantitative analyte evaluation usinga cut-off algorithm, 172.

The algorithm then determines whether the test is a clinical test, anexternal control or a calibration test, 174, and if the determination isyes (based on information provided on the test device bar code or basedon user input information), the results are stored to memory, 176, suchas on the SD card or in the apparatus memory. If the test is not aclinical test, then results are stored to flash memory, 180, anddisplayed and/or printed, 182. The drawer is then opened, 184, by theapparatus or by the user at the end of the measurement sequence for theuser to remove the test device, 186.

It will be appreciated that the test sequence is easily varied by simplyvarying the programming in the software programs in the device, to alterthe sequence of events, time allocated to each event, etc., in ameasurement procedure.

The measurement sequence by the optics system can include activating themotor that moves the optics module, and the optics module finding itshome position. At a first position along the optical read path thatcorresponds with the test window on the test device inserted in theapparatus, the illumination source in the optics module is turned on andthen off, and during the off period fluorescent emission is detected bythe photodetector in the optics module. The detected emission is storedin memory, and the motor in the optics system advances the optics modulea fixed amount to its next position. The path the optics system followspermits excitation and emission of the normalization target, directly orindirectly, and of the test device. After completion of a predefinednumber of incremental steps along the length of the optical pathway andcapture of light emission at each step, the optics module is returned toits home position by the motor, and the motor is powered off. It will beappreciated from this description, that in some embodiments, theapparatus comprises a dynamic optics module of an illumination sourceand a photodetector, wherein the module is static during anillumination/detection sequence and resumes dynamic movement thereafter.It will also be appreciated that the dark reading, i.e, detectedemission during the off, or dark period, of the illumination-detectionsequence, is utilized for purposes of baseline and background and notfor time-resolved fluorescence.

The algorithm stored in apparatus memory for that particular assay thensearches the data array for the peak emissions for each of thenormalization target, the test line(s) and reference lines to calculateline intensities of peak area or peak height. The algorithm calculatesresults from the data array and stores the results to memory, such as onthe SD card inserted into the device. The calculated result can bedisplayed to the screen on the apparatus, or prompted to be printed bythe user, or stored in flash memory if needed. A user can then instructthe apparatus to open the drawer, to remove the test device, ending themeasurement procedure.

Assays and Analytes to be Detected

The system comprised of an apparatus and a test device as describedherein is intended for detection of any analyte of interest. Analytesassociated with a disorder or a contamination are contemplated,including biological and environmental analytes. Analytes include, butare not limited to, proteins, haptens, immunoglobulins, enzymes,hormones, polynucleotides, steroids, lipoproteins, drugs, bacterialantigens, viral antigens. With regard to bacterial and viral antigens,more generally referred to in the art as infections antigens, analytesof interest include Streptococcus, Influenza A, Influenza B, respiratorysyncytial virus (RSV), hepatitis A, B and/or C, pneumoccal, humanmetapneumovirus, and other infectious agents well known to those in theart.

In some embodiments, a test device intended for detection of one or moreof antigen associated with Lyme disease. In some embodiments, a testdevice designed for interaction with the apparatus is intended for usein the field of women's health. For example, test devices for detectionof one or more of fetal-fibronectin, chlamydia, human chorionicgonadotropin (hCG), hyperglycosylated chorionic gonadotropin, humanpapillomavirus (HPV), and the like, are contemplated. In anotherembodiment, a test device for detection of vitamin D is designed forinteraction with the apparatus and method of normalization describedherein.

The test devices are intended for receiving a wide variety of samples,including biological samples from human bodily fluids, including but notlimited to nasal secretions, nasopharyngeal secretions, saliva, mucous,urine, vaginal secretions, fecal samples, blood, etc.

The test devices, in some embodiments, are provided with a positivecontrol swab or sample. In some embodiments, a negative control swab orsample is provided. For assays requiring an external positive and/ornegative control, the apparatus is programmed to request a user toinsert into the apparatus a test device to which a positive controlsample or a negative control sample has been deposited. Kits providedwith the test device can also include any reagents, tubes, pipettes,swabs for use in conjunction with the test device.

III. Examples

The following examples are illustrative in nature and are in no wayintended to be limiting.

Example 1 Analysis of Test Device Utilizing a Normalization Target

Ten analyzers (instruments) were calibrated every day for 90 days, witha goal of reducing inter-analyzer and/or intra-analyzer variability bynormalizing RFU values on a scan-by-scan basis using an embeddedreference material (also known referred to as a “normalization target”)mounted on the movable drawer of the instrument that receives a testdevice.

The results of two studies, “Study 1” and “Study 2” are presented below,in which the mean percent deviation in RFU with no normalization targetand process, and mean percent range of RFU when including anormalization target and process for the ten analyzers is reported:

Study 1 Deviation +/− Normalized Range +/− Mean of 10 6.4% 2.2%instruments

Study 2 Deviation +/− Normalized Range +/− Mean of 10 5.5% 2.4%instruments

Without the use of a normalization target and procedure, the averageintra-analyzer variability of the ten analyzers was 6.4%, whereas whenthe analyzers were normalized according to the methods described hereinusing normalization target that was scanned concurrent with each testdevice scan, the average variability was decreased to 2.2%. Oneparticular instrument that had a 10% variability without a normalizationtarget, improved by a factor of four with per-scan normalization.

Overall, using the system and methods disclosed herein for per-scannormalization, analyzer variability was reduced by a factor greater thanabout 2.

Example 2 Analysis of Test Device Utilizing a Normalization Target

Forty test devices for detection of human chorionic gonadotropin (hCG)were provided, and the same concentration of the same sample (20 mIU/mL)was placed on each device. The test devices were read in an instrument,by initiating an optical scan of the test device and a normalizationtarget embedded in the optical path of the instrument. In each instancevariability in the detected signal (RFU) was compared with and withoutper scan normalization, and a coefficient of variation (CV) determined.

Normalized CV % CV % Reference Control (n = 40) 13.64% 12.58% hCG Line(n = 6) 12.30% 9.56%

Per-scan normalization reduced the coefficient of variation (CV) by 1%.

In summary, per-scan normalization reduces analyzer variability, andfalse positives are prevented with a significantly improved CV.

While a number of exemplary aspects and embodiments have been discussedabove, those of skill in the art will recognize certain modifications,permutations, additions and sub-combinations thereof. It is thereforeintended that the following appended claims and claims hereafterintroduced are interpreted to include all such modifications,permutations, additions and sub-combinations as are within their truespirit and scope.

It is claimed:
 1. A method to improve precision of a test result from aninstrument, comprising: providing an instrument comprising anormalization target embedded in or on the instrument, the normalizationtarget having an assigned normalization value based on its detectedsignal relative to detected signal associated with a calibration valuestored in instrument memory; scanning or imaging in a single scan: (i) atest cartridge for detection of vitamin D, the test cartridge insertedinto the instrument to obtain a test signal indicative of presence orabsence of vitamin D; and (ii) the embedded normalization target toobtain a current normalization value; and optionally adjusting the testsignal by a scaling factor based on the current normalization value,thereby obtaining a scaled test result with improved precision relativeto test signal.
 2. The method of claim 1, wherein the embeddednormalization target is affixed in an optical pathway in the instrument.3. The method of claim 2, wherein the embedded normalization target isaffixed to an element in the instrument that is capable of receiving atest strip.
 4. The method of claim 1, wherein the embedded normalizationtarget is affixed to an element in the instrument that is capable ofreceiving a test strip.
 5. The method of claim 1, wherein the scanningor imaging in a single scan comprises scanning by an optics module thatdetects emitted fluorescent light or emitted reflected light.
 6. Themethod of claim 1, wherein the scanning or imaging in a single scancomprises imaging using a digital camera.
 7. The method of claim 1,wherein the embedded normalization target is comprised of a rhodamine.8. The method of claim 7, wherein the rodamine is selected from thegroup consisting of rhodamine B, rhodamine 6G and sulphorhodamine G. 9.A method to improve precision of a test result from an instrument,comprising: providing an instrument comprising a normalization targetembedded in or on the instrument, the normalization target comprised ofa rhodamine and having an assigned normalization value based on itsdetected signal relative to detected signal associated with acalibration value stored in instrument memory; scanning or imaging in asingle scan: (i) a test cartridge inserted into the instrument to obtaina test signal, the test cartridge comprising reagents for detection ofvitamin D; and (ii) the embedded normalization target to obtain acurrent normalization value; and optionally adjusting the test signal bya scaling factor based on the current normalization value, therebyobtaining a scaled test result with improved precision relative to testsignal.
 10. The method of claim 9, wherein the embedded normalizationtarget is affixed in an optical field in the instrument.
 11. The methodof claim 9, wherein the embedded normalization target is affixed to anelement in the instrument that is capable of receiving a test strip. 12.The method of claim 9, wherein the scanning or imaging in a single scancomprises scanning by a movable optics module that detects emittedfluorescent light or emitted reflected light.
 13. The method of claim 9,wherein the scanning or imaging in a single scan comprises imaging usinga digital camera.
 14. The method of claim 9, wherein the rodamine isselected from the group consisting of rhodamine B, rhodamine 6G andsulphorhodamine G.
 15. A system, comprising: an instrument with anoptical field comprising an optics module or a digital camera positionedto interrogate a test device positionable in the optical field, and anormalization target embedded in or on the instrument directly orindirectly in the optical field, an external calibrant having acalibration value stored in instrument memory:
 16. The system of claim15, wherein the embedded normalization target and the external calibrantare comprised of the same material.
 17. The system of claim 15, whereinthe embedded normalization target and the external calibrant arecomprised of different materials.
 18. The system of claim 17, whereinthe embedded normalization target is comprised of a rhodamine.
 19. Thesystem of claim 18, wherein the rodamine is selected from the groupconsisting of rhodamine B, rhodamine 6G and sulphorhodamine G.
 20. Thesystem of claim 15, wherein the optical field comprises a movable opticsmodule.